Programmed cell death (PCD) is a selective genetically controlled process of cell deletion. This contrasts with cellular necrosis, a passive cellular injury that results in loss of membrane integrity and cell lysis. Wyllie, A.H. et al., Int. Rev. Cytology 68:251-306(1980). A cell in PCD can be identified by nuclear chromatin condensation, nuclear shrinkage, ordered cleavage of the nuclear DNA, compactness of cytoplasmic organelles, and the appearance of an irregular plasma membrane. Kerr, J. F. et al., Brit. J. Cancer 26:2392-57 (1972). A discrete change which occurs early, before nuclear alterations, is the redistribution of phosphatidylserine from the plasma membrane inner leaflet to become exposed at the cell surface. The execution of PCD is essential in the control of tissue shape and size (Alison, M. R. et al., J RCP 26:25-35 (1992)) and thus plays an important role during development of the nervous system. Deckwerth, T. L. et al., J. Cell Biol. 123:1207-1222 (1993).
PCD is a necessary component not only in the development of the nervous system (Lo, A. C. et al., Arch. Histol. Cytol. 58:139-139 (.1995)), but also in the pathophysiological conditions that lead to neurodegeneration. Neuronal PCD can be induced by a variety of toxic insults to the nervous system, such as cerebral ischemia (Hara, A. et al., Brain Res. 697:247-250 (1995)), excitotoxicity (Didier, M. et al., J. Neurosci. 16:2238-2250 (1996)), and nitric oxide (NO) exposure (Maiese, K., Clinical Neuropharm. 21:1-7 (1998); Palluy, O. et al., Neuroscience Letters 208:1-4 (1996); Vincent, A. M. et al., Exp. Cell. Res. in press (1998)). In some experimental systems, free radical NO induced PCD induction is both rapid and robust occurring within hours in at least 70% of the neuronal population. Vincent, A. M. et al., J. Neurosci. Res. 50:549-564 (1997). As a result of its ability to modulate neuronal survival, NO has become aggressively employed as a valuable tool to dissect the downstream cellular pathways that mediate PCD. Maiese, K. et al., J. Neurochem. 68:710-714 (1997); Maiese, K., Clinical Neuropharm. 21:1-7 (1998); Maiese, K. et al., Neurosci. Lett. 166:43-47 (1994); Vincent, A. M. et al., J. Neurosci. Res. 50:549-564 (1997).
The externalization of phosphatidylserine is known to occur very early during PCD. This phenomenon was first described in lymphocytes (Koopman, G. et al., Blood 84:1415-1420 (1994)), but has since been attributed to many murine and human cell types (Martin, S. J. et al., J. Exp. Med. 182:1545-1556 (1995)). A recent report has demonstrated that increased surface phosphatidylserine is an early marker of PCD in neuronal cell lines. Rimon, G. et al., J. Neurosci. Res. 48:563-570 (1997). Membrane asymmetry is believed to be maintained through the activity of aminophospholipid translocase. Vanags, D. M. et al., J. Biol. Chem. 271: 31075-31085 (1996). Phosphatidylserine externalization during PCD could result from reversal of aminophospholipid translocase activity, or loss of this enzyme coupled with random externalization of membrane phospholipids. Bratton, D. L. et al., J. Biol. Chem. 272:26159-26165 (1997). The presence of an unidentified "inside-outside phosphatidylserine translocase" also has been proposed. Allen, R. T. et al., J. Pharmacol Toxicol. Methods 37:215-228 (1997). Factors known to inhibit PCD, such as trophic factors (Maiese, K. et al., J. Cereb Blood Flow Metab. 15:440-449 (1995); Maiese, K. et al., J. Neurosci. 13:3034-3040 (1993)), metabotropic glutamate receptor activation (Maiese, K., Robertson J T, Nowak T S (eds). Armonk, N.Y.: Futura Publishing Company, Inc. 281-297 (1998); Maiese, K. et al., J. Neurochem. 66:2419-2428 (1996); Vincent, A. M. et al., J. Neurosci. Res. 50:549-564 (1997); Vincent, A. M. et al., Exp. Cell Res. in press (1998)), or benzothiazole administration (Maiese, K. et al., J. Neurochem. 68:710-714 (1997)), also can prevent phosphatidylserine exposure, suggesting that this event may be an integral part of the PCD pathway which occurs only after the cell has committed to die.
Since the majority of neuronal injury can occur through the induction of PCD, the ability to rapidly identify the onset and progression of PCD has become crucial to elucidate the multiple molecular mechanisms that modulate PCD. Current studies that characterize PCD rely on a variety of "fixed" assays to identify the end stages of PCD that include gel electrophoresis DNA fragmentation assays, DNA 3'-OH end labeling, electron microscopy, and hematoxylin and eosin staining. Hill, I. E. et al., Brain Res. 676:398-403 (1995); Maiese, K., Clinical Neuropharm. 21:1-7 (1998); Vincent, A. M. et al., J. Neurosci Res. 50:549-564 (1997). A recent study defined PCD sequentially in hippocampal neuronal cultures, using techniques which do not permit the later re-assessment of the neuronal population. Maiese, K., J. Neurochem. 68:710-714 (1997); Vincent, A. M. et al., Exp. Neurol. (in press) (1998); Vincent, A. M. et al., J. Neurosci Res. 50:549-564 (1997); Vincent, A. M. et al., Exp. Cell Res. (in press) (1998). Other assays have incorporated the use of annexin V, a member of a family of calcium-dependent phospholipid-binding proteins, that possesses strong affinity for phosphatidylserine. Andree, H. A. et al., J. Biol. Chem. 265:4923-4928 (1990); U.S. Pat. No. 5,834,196. Since phosphatidylserine translocation occurs early in PCD when cell membrane integrity is still intact, flow cytometric analysis using fluorescein isothiocyanate-labeled annexin V is now widely used as a quantitative measure of early PCD. Homburg, C. H. et al., Blood 85:532-540 (1995); Koopman, G. et al., Blood 84:1415-1420 (1994); Vanags, D. M. et al., J. Biol. Chem. 271:31075-31085 (1996). Yet, flow cytometric techniques have limited application to adherent cultures, since harvesting of the sample generates certain mechanical membrane damage, and the use of trypsin can artificially induce phosphatidylserine exposure. van Engeland, M. et al., Cytometry 24:131-139 (1996). Assays that eliminate cell harvesting continue to require cell fixation, i.e., non-viable cells. Rimon, G. et al., J. Neurosci. Res. 48:563-570 (1997). It would thus be desirable to provide a method for analyzing PCD in living cells with minimal disruption of the cells. It would also be desirable to provide a method for temporally analyzing PCD in living cells wherein the onset and subsequent progression of PCD may be observed.